Translocation of Sulfate in Soybean ( Glycine max L

MATERIALS AND METHODS Sulfate translocation in soybean (Glycine max L. Merr) was investigated. More than 90% of the sulfate entering the shoot system was recoverable in one or two developing trifoliate leaves. In young plants, the first trifoliate leaf contained between 10 to 20 times as much sulfate as the primary leaves, even though both types of leaf had similar rates of transpiration and photosynthesis. We conclude that most of the sulfate entering mature leaves is rapidly loaded into the phloem and translocated to sinks elsewhere in the plant. This loading was inhibited by carbonylcyanide m-chlorophenylhydrazone and selenate. At sulfate concentrations below 0.1 millimolar, more than 95% of the sulfate entering primary leaves was exported. At higher concentrations the rate of export increased but so did the amount of sulfate remaining in the leaves. Removal of the first trifoliate leaf increased two-fold the transport of sulfate to the apex, indicating that these are competing sinks for sulfate translocated from the primary leaves. The small amount of sulfate transported into the mesophyll cells of primary leaves is a result of feedback regulation by the intracellular sulfate pool, not a consequence of their metabolic inactivity. For example, treatment of plants with 2 millimolar aminotriazole caused a 700 nanomoles per gram fresh weight increase in the glutathione content of primary leaves, but had no effect on sulfate aquisition. In a previous study of sulfate transport into soybean plants, we showed that more than 90% of the newly tansported sulfate in the shoot system was localized in a single developing leaf (16). Since the rate of transpiration of mature and developing leaves is not markedly different, similar amounts of sulfate must initially be drawn into both kinds of leaf. The ultimate accumulation of most of the sulfur in the developing leaf must therefore require translocation of sulfur. Several studies, with a variety of plants, show that sulfur is translocated in phloem as inorganic sulfate or sulfite (after exposure of plants to SO2 [7]) or as organic reduced sulfur, principally glutathione (3, 12). Biddulph et al. (2) suggested that the differential accumulation of sulfur in developing leaves was due to sulfate metabolism and incorporation of sulfur into amino acids and protein. However, the idea that immobilization of sulfur in a particular plant organ requires metabolic conversion to immobile molecules, such as proteins and sulfolipids, is not valid. For instance, when wheat plants are transferred from [35S]sulfate to unlabeled sulfate, the transport of label to the shoot stops immediately, even though 75% of the label in the roots is still present as sulfate (9). Our objective was to characterize sulfur translocation in soybean, with particular emphasis on the form in which sulfur is translocated and the explanation of its final distribution. ' Supported by National Science Foundation grant DMB 8421065 to I. K. S. Plants and Experimental Protocol. Soybean (Glycine max [L.] Merr) plants, raised from seed were grown in potting soil in a growth chamber, on a regime of 12 h light, intensity 400 gmol m-sI at 27°C and 12 h dark at 21°C. Plants were harvested, the soil was washed from the roots, and the plants were placed overnight in an aerated solution composed of 6 mM KNO3, 4 mM Ca(NO)2,, 1 mm MgCl,, and 2 mm (NH4)2HPO,, adjusted to pH 6.0 (25 plants/500 ml). Four intact plants were placed in 200 ml of the above medium suplemented with Na,[35S]O0 and exposed to light (400 ,umol m-) s ') at 27°C. Because sodium selenate and CCCP2 inhibit sulfate transport into roots, experiments with these compounds were done using shoot systems from which the roots were excised just prior to the experiment. CCCP was dissolved in 95% (vlv) ethyl alcohol and 0.2 ml added to 200 ml of sulfate-containing medium. When aminotriazole was used to stimulte glutathione accumulation, the light intensity was increased to 700 ,umol m sDetermination of Radioactivity. Discs (1.5 cm diameter) were cut from the lamina of the leaf with a cork borer, placed in scintillation vials with 1 ml 2% NaOCl (40% v/v commercial bleach), and the cuticle broken by tapping gently with a metal rod (18). Tissue was completely bleached and partially digested by incubating at 60°C for 1 to 4 h. Ammonium hydroxide (0.2 ml 4 M) was added to neutralize the sodium hypochlorite, and after 30 min at room temperature 10 ml ScintiVerse E (Fisher Scientific Co.) liquid scintillation cocktail was added. Samples were kept in the dark for 2 h before counting in a Beckman LS 3801 liquid scintillation counter. Transpiration. Rates of transpiration and CO, fixation were measured using an ADC model LCA2 IR gas analyzer and a PLC-N Parkinson Leaf Chamber (P. K. Morgan Instruments, Dallas, TX). The air flow was adjusted to 250 ml min ', light intensity was 300 ,umol m2 sl, ambient CO2 ranged from 345 to 370 ppm, and the temperature was 25°C. Duplicate measurements were taken from 10 plants at 11 AM, 1 PM, and 3 PM. Other Assays. Leaf tissue was homogenized in 5 volumes 5% (wlv) sulfosalicylic acid with a small amount of sand, a further 5 volumes of sulfosalicylic acid were added, and the brei centrifuged at 10OOg for 10 min to sediment insoluble material. GSH in the supernatant was measured by the coupled GSH reductase assay (15). Sulfate and sulfur amino acids present in the supernatant were separated by cation exchange chromatography. A 5 ml aliquot of the supernatant was passed through a Dowex 50 H+-form column (3.5 x 0.8 cm), and the column washed twice with 5 ml distilled water. Amino acids were eluted with 2 x 5 ml 3 M NH4OH. Radioactivity present in these samples was determined following the addition of 10 ml ScintiVerse E to 5 ml of the effluent or eluate. 'Abbreviation: CCCP, carbonylcyanide m-chlorophenylhydrazone. 798 www.plantphysiol.org on July 21, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. SULFATE TRANSLOCATION Table I. Distributioni of Newly Transported Sulfur in Soybean Planits of Different Ages Plants were placed in a medium containing 0.1 mm Na [35SJO4 (3.7 x 104 Bq/,amol) for 24 h. Values represent the means of duplicate samples